Shoot Formation from Leaf Explants and Apical Meristems of "Peperomia obtusifolia" A. Dietr. : Stability of Ploidy Level in Their Subcultures.

Bacterial soft rot can be a destructive disease of ornamental plants. To identify the pathogenic soft rot bacteria occurring in ornamental plants in Tehran and Markazi provinces (Iran), 57 isolates were obtained from 12 different mono- and dicotyledonous hosts and investigated with regard to phenotypic, genotypic and pathogenicity features. Based on phenotypic characteristics, the bacterial strains were identified as Pectobacterium carotovorum subsp. carotovorum. This was confirmed by subspecies-specific primers using PCR. Inoculation of all isolates into Aglaonema leaves confirmed the pathogenecity of the isolated strains. To assess the genetic diversity within Pectobacterium carotovorum subsp. carotovorum populations by rep-PCR, 26 isolates were selected according to their host and geographic distribution as well as two strains from Guilan province. Cluster analysis was conducted using the UPGMA method and revealed a possible close relationship between DNA fingerprints and geographical origins of isolates. Our results showed significant genetic variation among the populations of this pathogen. To the best of our knowledge, this is the first report of Pectobacterium carotovorum subsp. carotovorum on Peperomia obtusifolia, P. caperata, Pilea cadierei, Plectranthus australis, Saintpaulia ionantha and Kalanchoe tubiflora in Iran.

Labisia pumila var. alata, commonly known as 'Kacip Fatimah' or 'Selusuh Fatimah' in Southeast Asia, is traditionally used by members of the Malay community because of its post-partum medicinal properties. Its various pharmaceutical applications cause an excessive harvesting and lead to serious shortage in natural habitat. Thus, this in vitro propagation study investigated the effects of different plant growth regulators (PGRs) on in vitro leaf and stem explants of L. pumila. The capabilities of callus, shoot, and root formation were evaluated by culturing both explants on Murashige and Skoog (MS) medium supplemented with various PGRs at the concentrations of 0, 1, 3, 5, and 7 mg/L. Medium supplemented with 3 mg/L indole-3-butyric acid (IBA) showed the optimal callogenesis from both leaf and stem explants with (72.34 ± 19.55)% and (70.40 ± 14.14)% efficacy, respectively. IBA was also found to be the most efficient PGR for root induction. A total of (50.00 ± 7.07)% and (77.78 ± 16.47)% of root formation were obtained from the in vitro stem and leaf explants after being cultured for (26.5 ± 5.0) and (30.0 ± 8.5) d in the medium supplemented with 1 and 3 mg/L of IBA, respectively. Shoot formation was only observed in stem explant, with the maximum percentage of formation ((100.00 ± 0.00)%) that was obtained in 1 mg/L zeatin after (11.0 ± 2.8) d of culture. Callus, roots, and shoots can be induced from in vitro leaf and stem explants of L. pumila through the manipulation of types and concentrations of PGRs.

Jatropha curcas is a non-edible oil plant species and belongs to Euphorbiaceae family. It is gaining the importance as potential renewable feed-stock for biodiesel production. Here in the present study we developed an efficient micropropagation protocol through somatic embryogenesis from leaf and shoot tip explants of Jatropha curcas . Murashigae-Skoog medium supplemented with 2, 4-D (0.5mg/l) and benzylaminopurine (5mg/l) resulted in the production of green embryogenic callus with clearly differentiated somatic embryos with a frequency of plant conversion rate from shoot tip and leaf explants was 51 ± 0.9 and 54 ± 0.6 respectively. The regenerated shoots were rooted on root induction medium, transplanted to pots and later transferred to field. The survival percentage of the transplanted plants was 33±0.1 for shoot tip and 24±0.7 for leaf explant. Therefore, this protocol could be useful for micropropagation of Jatropha curcas as effective feed-stock towards biodiesel production.

In vitro adventitious shoot formation was initiated from bulb and leaf explants of Haemanthus deformis, H. humilis, and H. albiflos. The initiation medium used was Murashige and Skoog (MS) without plant growth regulators, although shoots and callus were later subcultured onto MS medium containing 2 mg 1−1 kinetin and 1 mg 1−1 IAA, which stimulated shoot and root development, especially from the callus. H. humilis was the first species to initiate shoots from bulb explants, with 76% direct organogenesis. H. deformis had 20% shoot and root formation from bulb explants and 30% shoot formation from leaf explants. H. albiflos produced 50% shoot formation from leaf explants and the callus developed numerous shoots once placed on medium with growth regulators. On average it took 18–20 weeks from shoot initiation to obtain plantlets ready to be acclimatised. Leaf explants in general took longer to initiate shoots, although they produced more shoots per explant, and also had less contamination compared to the bulb explants. Plantlets with roots were successfully acclimatised in vermiculite under misthouse conditions, and hardened off in a 70% shadehouse. Keywords· Bulb and leaf explants, Haemanthus, micropropagation, shoot formation, conservation.

Production of genetically and phenotypically stable plantlets is the main purpose in commercial strawberry tissue culture. In this study, different tissues of Fragaria ananassa cv. Camarosa including stipule, apical meristem, leaf and petiole were cultured in Murashige and Skoog (MS) medium supplemented with different concentrations of N6-benzyladenine (BA) (0.5, 1, 2, 3 mg/L) and indole-3-butyric acid (IBA) (0.1 and 0.5 mg/L). For apical meristem explants, the best regeneration rate (7.6 shoots per each explant) was obtained in the medium containing 1 mg/L BA and 0.1 mg/L IBA. Whereas stipule explants showed the highest regeneration rate in the medium containing 2 mg/L BA and 0.1 mg/L IBA. For leaf and petiole explants, the medium containing 2 mg/L BA and 0.5 mg/L IBA had the best hormonal combination. To determine the genetic variation, micropropagated plants from different tissue (after 8 subcultures) were analyzed by the inter-simple sequence repeats (ISSR) molecular marker. Among the 18 pre-selected primers, 10 displayed clear, reproducible and informative bands. A total of 88 distinct bands with a polymorphic rate of 46% were produced in the molecular profile of different explants. The highest and the lowest similarity values to maternal plants belonged to stipule and petiole explants with a similarity index of 0.738 and 0.645, respectively. Vitroplants derived from stipule and apical meristem showed the highest genetic stability with respect to maternal plants. With respect to genetic stability and regeneration rate, apical meristem can be recommended as suitable explants with the highest genetic fidelity. The findings of this study could be applied for commercial scale multiplication of strawberry, and also demonstrate that ISSR markers are eligible for detection of somaclonal variations.

The objectives of this study were to develop a tissue culture system for mass propagation of tuberous begonia (Begonia x tuberhybrida Voss). Adventitious shoots were successfully regenerated from leaf or petiole explants of hybrid tuberous begonia (Begonia x tuberhybrida Voss). Leaf and petiole explants were cultured on MS medium with NAA (0.54 or 5.4 μM) and BA (0.44, 2.22, 4.4 or 22.2 μM). Adventitious shoots were successfully regenerated and the optimum medium for adventitious shoot induction of leaf or petiole explants was MS medium supplemented with 5.4 μM NAA and 2.22 μM BA both for leaf and petiole explants, with adventitious shoot induction of 1.3 and 5.0, respectively. The adventitious shoots with original tissues were transferred to liquid MS with 3.0 g L^(-1) activated charcoal cultured for 4 weeks and then 20.3 plantlets were regenerated. After rooting and the acclimatization process, plantlets were transplanted to the greenhouse. The survival rate of regenerated plantlets was 65%. From those results, we devised an adventitious shoot regeneration system from leaf explants for rapid and efficient proliferation of tuberous begonia.

Functional morphology and productivity of a tussock grassland in the Bolivian Altiplano

Adventitious shoot formation was studied with leaf, stem and axillary bud explants of carnation (Dianthus caryophyllus L.). The shoot regeneration procedures were applicable for a wide range of cultivars and shoot regeneration percentages were high for all explant types. Using axillary bud explants, shoot regeneration efficiency was independent of the size of the bud and of its original position in the plant. In contrast, shoot regeneration from stem and leaf explants was strongly dependent on their original position on the plant. The most distal explants (just below the apex) showed the highest level of shoot regeneration. The adventitious shoot primordia developed at the periphery of the stem segment and at the base of leaf explants. In axillary bud, stem and leaf explants, shoot regeneration originated from node cells, located at the transition area between leaf and stem tissue. Moreover, a gradient in shoot regeneration response was observed, increasing towards the apical meristem.

Shallot latent virus (SLV) was detected by DTBIA, and evenly distributed in infected bunching onion (Allium fistulosum L.). An ELISA procedure was efficiently assigned for routine virus indexing in healthy seedling mass production system. For culture in MS medium, tissues from SLV infected bunching onion were excised, and the tested explants including stem-disc, apical meristem, meristem with one primordial leaf, and meristem with two primordial leaves. The results showed that only the plantlets derived from apical meristem are SLV virus-free. The rapid multiplication of healthy stock was also established. It was demonstrated that the number of regenerated shoots from stem-disc was reduced when TDZ and NAA combined together in MS media, but TDZ alone would regenerate more shoots. The stem-disc treated with 1-5 mg/L TDZ for 42 days, the optimal number of shoots (43-62) were regenerated. The TDZ had better effect on induction of shoot regeneration than BA or kinetin.

Coleoptile, leaf and root explants of the einkorn (Triticum monococcum ssp. monococcum) were cultured in vitro to obtain an efficient plant regeneration protocol through direct shoot formation by using different combination and concentration of various plant growth regulators. A total of 180 different auxin and cytokinin combinations were tested for regeneration. Shoot formation was not observed with the root and leaf explants. Shoot formation was obtained only from the coleoptile explants, with a mean of 1.20 ± 0.24 shoots/explant and 86.60% of shoot formation frequency and with a 1.20 ± 0.53 shoots/explant and 80.00% shoot formation frequency on medium supplemented with 0.5 mg L-1 TDZ and 1 mg L-1 TDZ plus 1 mg L-1 NAA, respectively. The shoots were subculture on the MS medium containing the most effective hormone combination concurrently continued to shoot and root formation for 45 days. It was noteworthy that 3.66 ± 0.66 shoots per explant were induced by MS, which contained 1 mg L-1 TDZ plus 1 mg L-1 NAA and 2.0 mg L-1 KIN plus 0.5 mg L-1 NAA during a 45 days. Of the different auxin concentrations tested for rooting, 2.0 mg L-1 IAA was predominant, with the greatest number of roots (12.33 ± 0.88) produced per regenerated shoot. Finally, these well-developed plantlets were acclimatized with a 100 % success rate and they transferred to the ex vitro conditions. A highly efficient regeneration protocol for einkorn wheat was developed using somatic tissue as an explant source for the first time.

The purpose of the study was to determinethe effect of colchicine on morphology and number of chromosome of binahong plant ( Anredera cordifolia Tenn. Steenis.). The research was conducted at Faculty of Agriculture USUMedan, with a height of 25 metre above sea level, from May until December 2015. The design of the research was a Randomized Block Design of one factor with 5 treatments were: Control (without treatments);0.025% ;0.050%;0.075%;0.100%. Parameters observed were number of leave, weight of fresh shoot per sample, weight of dry shoot per sample, weight of fresh root, weight of dry root and number of chromosome. The results showed that the effect colchicine gave significantly to number of leave, weight of fresh shoot per sample, weight of dry shoot per sample, number of chromosome : 2n=2x+1=23 set (0.025%), 2n=2x+1=23 set (0.050%), 2n=2x+2=24 set (0.075%) and 2n=2x+4= 26 set (0.100%) with normally chromosome 2n=2x=22 set. Key words : colchicine, morphology, number of chromosome, binahong plant

Higher plants maintain continuous development throughout their life by closely regulating the process of cell differentiation (Clark, 2001; Sablowski, 2007). In plants, the balance between undifferentiated and differentiated cell fate is managed within a stem cell niche termed the meristem. Cell differentiation in the meristem is in part controlled by genetic mechanisms. For example, mutations in CLAVATA (CLV) genes increase the number of undifferentiated cells within shoot and floral meristems leading to supernumerary organs (Clark, 2001). In contrast, mutations in genes of the homeodomain transcription factors WUSCHEL (WUS) and SHOOT-MERISTEMLESS (STM) lead to the absence of the shoot or floral meristem or its early termination through differentiation (Laux et al., 1996; Long et al., 1996). Cell differentiation in the meristem is also controlled by hormonal cues, which interfaces with gene function. For example, cytokinin treatment leads to phenotypes resembling clv mutants (Lindsay et al., 2006). Furthermore, exogenous cytokinin treatment has been shown to rescue the stm mutant phenotype and WUS protein has been shown to repress transcription of genes that act in the negative feedback pathway of cytokinin signaling (Leibfried et al., 2005; Yanai et al., 2005). The plant hormone auxin also plays a role in regulating differentiation. Auxin is thought to stimulate the initiation, development and differentiation of cells specified into organs (Teale et al., 2006). Disruption of auxin transport leads to a reduction in organ initiation and differentiation (Okada et al., 1991). In this thesis we investigate spatially regulated signaling and action of auxin and cytokinin which regulate patterning of gene expression and cell differentiation. To this end, we employed two model systems of shoot meristem initiation and development in the model plant Arabidopsis thaliana: shoot and floral meristem development and de novo shoot meristem initiation from tissue culture. Based on characterization of hormone signaling and patterning of gene expression during de novo shoot meristem initiation from tissue culture we propose a novel Turing-like model by which auxin and cytokinin interact to regulate patterning of cell differentiation. In this model, the activity of auxin, the activator of cell differentiation, is regulated by cytokinin, an inhibitor of cell differentiation. Computational models of these interactions lead to self organizing patterning of hormone response and cell differentiation as observed in experiments. In our second investigation, we show that cytokinin signaling regulates the spatial patterning of the homeodomain transcription factor WUS within the shoot meristem. We demonstrate that WUS misregulation after cytokinin treatment is mediated by both CLAVATA-dependent and independent mechanisms leading to multiple feedback loops. We reveal the presence of a cytokinin perception and signaling gradient within the shoot meristem, which spatially influences size and position of the WUS domain. Finally, we have begun to identify the molecular components required for cytokinin activation of WUS expression. Of the three characterized cytokinin receptors, only Arabidopsis Histidine Kinase 2 (AHK2) is required for WUS induction in the presence of cytokinin. In contrast, the AHK3 receptor is required for negative feedback on cytokinin signaling and thus WUS. These data reveal an unappreciated specificity in cytokinin signaling in regulating downstream targets which may be important for eliciting different cell behaviors depending on the threshold of signaling and the ratio of the three cytokinin receptors within a given cell.

ストックの本葉および胚軸からのカルスおよび器官形成(シュート形成)に及ぼす供試植物の栽培条件(光条件•施肥条件)ならびに培地中の窒素組成の影響について検討した.1.葉片培養:(1)光および施肥条件の影響;'銀潮'では遮光処理(75%遮光)によリシュート形成が促進された.また遮光の効果は1/2倍水耕液を施用した区で顕著であった.一方,'雪まつり'では1/4および1/2倍液施用区とも遮光処理によってシュート形成は抑制された.(2)培地の窒素組成の影響;'銀潮'および'雪まつり'とも,アンモニア態窒素無添加区(硝酸態窒素のみ)において高いシュート形成率を示し,NH4++/NO3-比を高めるとシュート形成が抑制される傾向を示した.2.下胚軸培養:(1)光および施肥条件の影響;'リトルゼムイエロー'および'雪まつり'とも,遮光処理によってシュート形成が抑制された.また,外植体当たりのシュート数も遮光処理によって減少した.(2)培地の窒素組成の影響は,'リトルゼムイエロー'および'雪まつり'とも,アンモニア態窒素の添加が,シュート形成率および外植体当たりのシュート数を高める傾向を示した.'リトルゼムイエロー'ではNH4++:NO3-が1:10の培地で,また'雪まつり'ではNH4++:NO3-が1:2の培地でそれぞれ85%と最も高い形成率を示した.3.形成されたシュートは,大部分が水浸状を呈していた.水浸状の葉は,水分を含んで肥大し,その組織では細胞間隙が大きく,維管束や棚状組織の発達が不十分であった.また気孔の分布が一様ではなく,その数も正常葉と比較して少なく,孔辺細胞が表皮細胞より陥没しているものや隆起しているものなどが観察された.

Thirteen cassava cultivars were collected from farmers in the Greater Accra Region using a structured questionanire. Five cultivars namely, Ankrah, Bosom nsia, Biafra, Santom and Afisiafi were selected based on popularity, duration to maturity and tolerance to African Cassava Mosaic Virus (ACMV) disease. The cultivars were propagated in vitro using meristem, multiple shoots culture, and somatic embryogenesis. Meristematic explants were cultured on Murashige and Skoog (1962) basal salts and vitamins (MS) amended with NAA 0.1 mg/l, GA3 0.1 mg/l BA (0.0-0.15 mg/l BA). There was profuse callus formation in all the cultivars. The optimal concentration for shoot proliferation was 0.10 mg/l BA or 0.15 mg/l BA. With reduced NAA and GA3 concentrations (0.02 and 0.04 mg/l respectively) in the culture medium BA 0.05 mg/l was optimum with 100% and 46% shoot regeneration respectively in Bosom nsia and Santom compared to 37% and 0% in the ptevious treatment. All the selected cultivars formed multiple shoots from single bud cutting of in vitro plantlets. However, the number of apical shoots formed was dependent on BA concentration in the medium. Embryogenic calli formation on MS amended 2,4-D 0.0-16 mg/l depended on the type of explants. For greenhouse grown plants development of embryogenic calli from young leaf lobe and apical meristem explants was significantly higher than stipule explants. However, none of the calli were able to induce primary embryos when transferred to a maturation medium (MS plus 0.1 mg/l BA). Similarly embryogenic calli formation from tissue-cultured young leaf lobe explants on the same media were high in all the 2,4-D treatments. Subsequent production of primary embryo was low on the maturation medium and was found to depend on the cultivar and 2,4-D concentration. Somatic embryo formation was higher on 2,4-D 16 mg/l medium than on 4 mg/l or 8 mg/l 2,4-D medium. Santom produced the highest percentage of embryo (25%) among the cultivars used. Embryogenic calli which did not form somatic embryos formed roots which depended on the 2,4-D concentration of the induction medium. JOURNAL OF THE GHANA SCIENCE ASSOCIATION Volume 1 Number 3, July (1999) pp. 31-41

The diversity of olive (Olea europaea L.) germplasm is under significant threat in its traditional area of cultivation. Clonal field collections are the usual means of preserving olive germplasm. Cryopreservation, the preservation of tissues and organs in liquid nitrogen at -196°C, offers a potential complementary approach that overcomes some of the problems associated with field collections. Here, recent advances in the application of cryogenic techniques to olive germplasm are discussed. For cryopreservation, shoot tips (i.e. the apical meristems, surrounded by leaf primordia and excised from in vitro shoot cultures) are the best explants to assure genetic fidelity to the donor plant. Olive shoot tips have been cryopreserved using PVS2 vitrification or osmoprotection/dehydration approaches. Although up to 30% of post-thaw survival has been reported, shoot tip regrowth into plantlets was always problematic. Unlike shoot tips, cryopreservation of embryogenic tissue and somatic embryos of olive have been successful using a variety of controlled rate and vitrification approaches. Although less suited to germplasm conservation as compared with shoot tips, the ability to cryopreserve olive embryogenic cultures has important implications for bioengineering, e.g. avoiding the loss of embryogenic potential due to repeated subculturing or allowing the storage of transgenic material while field trials are ongoing. Potential approaches towards the improvement of olive cryostorage, which will hopefully allow the future development of germplasm cryo-banks, are discussed in terms of current developments in woody plant cryopreservation.